The RNeasy MinElute Cleanup Kit enables cleanup and concentration of RNA from enzymatic reactions or other samples using specialized RNeasy MinElute spin columns based on silica-membrane technology. The kit can also be used to desalt RNA samples. Up to 45 μg RNA can be purified in a volume as low as 10 μl. Purification can be fully automated on the QIAcube Connect.

The RNeasy MinElute Cleanup Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

High-quality RNA.

Total RNA was isolated from HeLa cells using an acid-phenol method plus cleanup and concentration using the RNeasy MinElute Cleanup Kit. The high quality of the RNA is shown by scanning with the Agilent 2100 Bioanalyzer.

Concentration of RNA.

The indicated amounts of total RNA from HeLa cells were concentrated using the RNeasy MinElute Cleanup Kit, and 5 µl of the 12 µl eluate was used for each reaction. Real-time RT-PCR was carried out on an ABI Sequence Detection System using the QuantiTect Probe RT-PCR Kit, with primers and probes specific for β-actin.

Reliable cleanup of RNA.

The indicated amounts of total RNA from HeLa cells were concentrated using the RNeasy MinElute Cleanup Kit. Real-time RT-PCR was carried out on the LightCycler System using the QuantiTect Probe RT-PCR Kit, with primers and probes specific for the p16 gene.

Efficient removal of real-time RT-PCR inhibitors.

Without cleanup, inhibitors can lead to underestimation of RNA amounts in real-time quantification. Total RNA was isolated from HeLa cells using an acid-phenol method, with or without subsequent cleanup using the RNeasy MinElute Cleanup Kit. Real-time RT-PCR was carried out as described in figure "Concentration of RNA", with the indicated volumes of the RNA eluate. Apparent RNA amounts were calculated from the resulting CT values.

Performance

The RNeasy MinElute Cleanup Kit provides high-quality total RNA, free from impurities or enzymatic inhibitors, with A260/A280 ratios of 1.9–2.1 (see figure "High-quality RNA"). RNA amounts corresponding to less than one cell (as little as 1 pg) can be concentrated (see figure "Concentration of RNA"). A large amount of RNA (up to 45 µg) can be purified and is suitable for use in sensitive assays (see figure "Reliable cleanup of RNA"). Reaction volumes can be kept small in downstream applications, giving increased reaction efficiency. The high purity RNA allows more of the sample to be used in reactions without inhibition (see figure "Efficient removal of real-time RT-PCR inhibitors").

Principle

The RNeasy MinElute Cleanup Kit is designed to purify and concentrate RNA from enzymatic reactions (e.g., labeling, in vitro transcription), RNA isolated by alcohol-precipitation and organic-extraction methods, as well as to desalt RNA samples, and concentrate RNA prepared by silica-membrane procedures (e.g., PAXgene Blood RNA preps). RNeasy technology simplifies total RNA isolation by combining guanidine-isothiocyanate lysis with silica-membrane purification.

Procedure

Guanidine-isothiocyanate–containing lysis buffer and ethanol are added to the sample to create conditions that promote selective binding of RNA to the RNeasy MinElute membrane. The sample is then applied to the RNeasy MinElute spin column. RNA binds to the silica-membrane, contaminants are efficiently washed away, and high-quality RNA is eluted in water.

Enzymatic reactions or crude RNA preps are simultaneously cleaned up and concentrated in less than 15 minutes. In comparison, time-consuming concentration and cleanup by alcohol precipitation can result in loss of RNA, especially from small samples. The RNeasy MinElute procedure is faster than vacuum centrifugation, which only concentrates the sample without removing salts and other impurities. The unique design of RNeasy MinElute spin columns enables concentration of purified RNA to as little as 10 µl for downstream applications such as microarray analysis and real-time RT-PCR. Purification can be fully automated on the QIAcube.

Applications

RNA purified with RNeasy MinElute technology is high-quality and ideal for use in all applications including:

There are two protocols: follow Protocol 1 if you want to purify total RNA containing miRNA, or follow Protocol 2 if you want to purify small RNA (includes miRNA, 5S rRNA, and tRNA) and larger RNA (>200 nt) separately.